Mitochondrial import of cleavable preproteins occurs at translocation contact sites, where the translocase of the outer membrane (TOM) associates with the presequence translocase of the inner membrane (TIM23) in a supercomplex. Different views exist on the mechanism of how TIM23 mediates preprotein sorting to either the matrix or inner membrane. On the one hand, two TIM23 forms were proposed, a matrix transport form containing the presequence translocase-associated motor (PAM; TIM23-PAM) and a sorting form containing Tim21 (TIM23 SORT ). On the other hand, it was reported that TIM23 and PAM are permanently associated in a single-entity translocase. We have accumulated distinct transport intermediates of preproteins to analyze the translocases in their active, preprotein-carrying state. We identified two different forms of active TOM-TIM23 supercomplexes, TOM-TIM23 SORT and TOM-TIM23-PAM. These two supercomplexes do not represent separate pathways but are in dynamic exchange during preprotein translocation and sorting. Depending on the signals of the preproteins, switches between the different forms of supercomplex and TIM23 are required for the completion of preprotein import.The majority of mitochondrial proteins are nuclear encoded and posttranslationally transported into the organelle. A major class of mitochondrial proteins possess cleavable targeting signals at their amino termini, so-called presequences (5,9,12,19,30,32). These ␣-helical segments are positively charged and direct the proteins across the outer and inner mitochondrial membranes toward the matrix space, where the presequences are proteolytically removed. However, a number of proteins of the inner mitochondrial membrane, among them subunits of the respiratory chain complexes, also utilize presequences as targeting signals. In addition to the presequence, they contain a hydrophobic sorting signal, which arrests precursor translocation across the inner membrane and mediates the lateral release of the polypeptide into the lipid phase (16,30). In some cases, the membrane-inserted precursors undergo a second processing event by the inner membrane protease that cleaves behind the sorting signal and therefore leads to the release of the protein into the intermembrane space (25, 30, 31). Thus, a large variety of proteins destined for three different intramitochondrial compartments use presequences as the primary signal for transport.Cleavable preproteins initially enter mitochondria via the TOM complex and are translocated into or across the inner membrane by the TIM23 complex. The TIM23 complex consists of four integral membrane proteins, Tim23, Tim17, Tim50, and Tim21. Tim23 forms the protein-conducting channel of the translocase and is tightly associated with Tim17 (8,26,43). Tim50 acts as a regulator for the Tim23 channel and is involved in early steps of precursor transfer from the outer to the inner membranes (23,29,41). Tim21 transiently interacts with the TOM complex via binding to the intermembrane space domain of Tom22. This interaction prom...

Mgr2 is a new component of the mitochondrial presequence translocase required for efficient coupling of the TIM23 core complex to Tim21, respiratory chain complexes, and the translocase of the outer mitochondrial membrane.

The presequence translocase of the inner mitochondrial membrane (TIM23 complex) is essential for importing cleavable preproteins into mitochondria. The preproteins contain amino-terminal targeting sequences that are removed by the mitochondrial processing peptidase (MPP). Some preproteins carry bipartite presequences that are cleaved twice, by MPP and the inner membrane protease (IMP). Here, we report that the TIM23 complex is altered in mitochondria lacking the IMP subunit Imp1 although none of the TIM23 components contains a bipartite presequence. We show that the TIM23 subunit Mgr2 is processed by IMP, but not by MPP. The cytosolic precursor of Mgr2 contains a carboxy-terminal sequence that promotes targeting to mitochondria, but impairs stable assembly and function of the mature TIM23 complex. IMP removes the carboxy-terminal targeting sequence and thus promotes proper assembly of the TIM23 complex. Our results reveal carboxy-terminal processing as a new mechanism in the biogenesis of the mitochondrial inner membrane.

Brugada syndrome (BrS) is one of the major causes of sudden cardiac death in young people, while the underlying mechanisms are not completely understood. Here, we investigated the pathophysiological phenotypes and mechanisms using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from two BrS patients (BrS-CMs) carrying a heterozygous SCN5A mutation p.S1812X. Compared to CMs derived from healthy controls (Ctrl-CMs), BrS-CMs displayed a 50% reduction of I Na density, a 69.5% reduction of Na V 1.5 expression, and the impaired localization of Na V 1.5 and connexin 43 (Cx43) at the cell surface. BrS-CMs exhibited reduced action potential (AP) upstroke velocity and conduction slowing. The I to in BrS-CMs was significantly augmented, and the I CaL window current probability was increased. Our data indicate that the electrophysiological mechanisms underlying arrhythmia in BrS-CMs may involve both depolarization and repolarization disorders. Cilostazol and milrinone showed dramatic inhibitions of I to in BrS-CMs and alleviated the arrhythmic activity, suggesting their therapeutic potential for BrS patients.

Background:The TIM23 machinery cooperates with partner protein complexes of the inner mitochondrial membrane to drive preprotein import. Results: The mitochondrial ADP/ATP carrier is efficiently copurified with TIM23 complexes independently of the respiratory chain. Conclusion:The ADP/ATP carrier is an integral part of the TIM23 protein network and supports preprotein import. Significance: This study reveals an unexpected link between mitochondrial protein biogenesis and metabolite transport.

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In order to evaluate the aclimation of Chenopodium seedlings to different quantum fluence rates of R and BL, kinetics of Rubisco capacity, Chl content and chloroplast structure were studied. Under monochromatic light photoreceptors are stimulated selectively and their influence on biosynthetis capacities during chloroplast development can be studied.R irradiations saturate Rubisco capacity even at the lowest quantum fluence rates applied, whereas Chl a+b synthesis depends strongly upon fluence rate of R. Under BL irradiations, both Rubisco capacity and Chl content are fluence rate dependent. R irradiations favour Chl b synthesis relative to Chl a, whereas under BL Chl a content is high relative to Chl b. Under R irradiation Pfr is the main photoreceptor involved in regulation of Rubisco capacity whereas under BL a specific BL absorbing photoreceptor may control the response. From the fluence rate dependency under BL irradiations it is concluded that the blue region of the day light spectrum may be the sensor for monitoring fluence rate and causing the characteristic changes in shade and high/low WL adaptation with respect to Rubisco levels in Chenopodium.

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